noroviruses (known as Norwalk-like viruses or Norwalk viruses) are enteropathogenic viruses that cause acute gastroenteritis in adults and children. Viruses belonging to the genera norovirus are responsible for over 90% of all non-bacterial gastroenteritis epidemics [1] and a leading cause of global diarrhea [2]. The high prevalence of norovirus infections has led investigators to develop vaccine candidates to prevent disease [3]. Norwalk virus (NV) is the prototype virus of the genera norovirus and extensive preclinical studies in mice have shown that NV virus-like particles (VLPs) administered parenterally, orally, or intranasally are immunogenic [3-9]. In clinical trials, NV VLPs administered orally or intranasally have been shown to be well tolerated and modestly immunogenic [10-12]. Despite promising results, many challenges to developing a norovirus vaccine remain. A key obstacle has been the incomplete understanding of the immune correlates of protection [3, 9, 13], although a recent publication by Reeck at al. showed that antibodies that block histoblood group antigen binding to NV VLPs correlate with protection against clinical NV gastroenteritis [14].
In developing countries, according to a 2008 estimate by CDC researchers, up to 200,000 children under 5 years old die of norovirus infection each year. There is no vaccine against norovirus and no specific antiviral drugs to treat infections.
Norwalk virus is a round, nonenveloped, ˜27-nm virion. Its nucleic acid contains single-stranded, positive-sense RNA. It has a single structural protein characteristic of a calicivirus. The single, positive strand of Norwalk virus RNA contains three open reading frames, the second of which is known to encode a single NV capsid protein (NVCP) that self-assembles into empty virus-like particles (VLPs) lacking viral RNA when expressed in the baculovirus/insect cell expression system. X-ray crystallography of recombinant NV VLPs (rNV VLPs) showed that these VLPs are composed of 90 dimers of the NVCP that form T=3 icosahedral structure with a diameter of about 38 nm.
The rNV VLPs are stable at low pH, when lyophilized, and when stored long term at 4° C. The insect cell-derived VLPs are immunogenic in experimental animals and in human volunteers following oral administration (Ball et al., “Oral Immunization with Recombinant Norwalk Virus-Like Particles Induces a Systemic and Mucosal Immune Response in Mice,” J Virol 72: 1345-1353 (1998), Ball et al., “Recombinant Norwalk Virus-Like Particles Given Orally to Volunteers: Phase I Study,” Gastroenterology 117: 40-48 (1999)), and in mice when administered parenterally (Jiang et al., “Expression, Self-Assembly, and Antigenicity of the Norwalk Virus Capsid Protein,” J Virol 66: 6527-6532 (1992)), and intranasally (Guerrero et al., “Recombinant Norwalk Virus-Like Particles Administered Intranasally to Mice Induce Systemic and Mucosal (Fecal and Vaginal) Immune Responses.,” J Virol 75: 9713-9722 (2001)). These qualities make the rNV VLPs useful as a candidate for vaccine against noroviruses, including Norwalk virus.
noroviruses cause greater than 90% of nonbacterial gastroenteritis outbreaks and an estimated 23 million cases of gastroenteritis in the U.S. per year. Although, the Norwalk strain of norovirus was the first discovered, it is now apparent that the Norwalk virus causes less than 10% of gasteroenteritis cases, whereas other members of the norovirus family, such as the Lordsdale virus, Toronto virus, Hawaii virus and Snow Mountain virus, may cause 90% of cases.
The symptoms of norovirus infection include simultaneous diarrhea and vomiting as well as fever, headaches, chills and stomach-aches. The cause of such symptoms may be related to the binding of noroviruses to carbohydrate receptors of intestinal epithelial cells, which results in an imbalance in ion transfer.
Extremely contagious, noroviruses can cause disease by infection with as few as 10 virions. Although, otherwise healthy people infected with noroviruses may recover within 2-4 days, they may still shed virus for up to 2 weeks after the onset of symptoms; hence, infected individuals should be quarantined for up to two weeks. Approximately 30-40% of infected people may remain symptom-free, though spread infection by shedding of virus to others who may be more susceptible to infection.
Recent estimates obtained by using new and improved diagnostic assays developed over the past decade for the detection of NV infections indicate that greater than 90% of outbreaks of acute nonbacterial gastroenteritis are caused by NV or Norwalk-like agents. Outbreaks frequently occur in day care centers, schools, nursing homes, hospitals, and the military. The increasing clinical significance of these infections suggests that an effective vaccine could be useful.
Most nonreplicating proteins administered alone by mucosal routes induce poor if measurable immune responses. Therefore, there remains a need for an improved therapy for treating patients having gastroenteritis associated with norovirus or sapovirus infection and methods for preventing the spread of infection.
The most effective means to prevent infectious diseases like norovirus is through vaccination strategies that initiate immune responses at the natural site of infection, the mucosa [15]. Currently, the majority of licensed vaccines are administered parenterally; however, these vaccines have the disadvantages of patient reluctance to tolerate needle sticks and lack of mucosal immune induction [16]. Previous studies have evaluated the immunogenic potential of oral, nasal, rectal, and vaginal routes of vaccine administration [17-28]. The nasal cavity is a promising site for vaccine delivery because it is easy to access, is highly vascularized, has a relatively large surface area, has low proteolytic activity, and is able to induce systemic immunity as well as both local and distal mucosal immunity via the Common Mucosal Immune System (CMIS) [16, 29-32]. An intranasal influenza vaccine was approved for clinical use by the U.S. Food and Drug Administration (FDA) [33-35] and other intranasal vaccines for hepatitis B virus (HBV), measles, anthrax, bacterial meningitis, and others are being evaluated [18, 36]. Additional VLP-based, nasal vaccines have been shown to induce distal mucosal and systemic immunity in mice [37, 38]. The nasal route has also been shown to be superior to parenteral administration for VLP-based vaccines at eliciting IgA at distal mucosal sites [39].
Nasally administered vaccines initiate an immune response through the nasal-associated lymphoid tissue (NALT) [32, 40]. The NALT is composed of an assembly of antigen-reactive cells including B cells, T cells, and antigen presenting cells (APCs). Upon nasal vaccine administration, antigens can be taken up by specialized epithelial cells called microfold cells (M cells), or by macrophages and dendritic cells, which in turn leads to the activation of T and B cells [40, 41]. Without an adjuvant or mucoadhesive, most nasal vaccines do not elicit an immune response due to the rapid clearance of antigens [32]. The addition of mucoadhesive compounds to the vaccine formulation increases the residence time in the nasal cavity, thus increasing antigen uptake by M cells and other APCs, and enhancing the immune response [30, 32, 42].